A new imaging method promises to simplify the process of determining antibiotic mechanisms of action. Learn more...

Several strategies are currently available for investigating mechanisms of
action during antibiotic drug discovery. The macromolecular synthesis
assay—which uses radioactive labeling to determine whether a compound blocks
RNA, DNA, lipid, or cell wall synthesis—is a common starting point, but it
falls short in

Schematic diagram of bacterial cytological profiling

terms of accuracy and throughput. Other ways to determine a drug’s mechanism
are to isolate either resistant or sensitized mutants or to use
transcriptional profiling. Each of these strategies has its own strengths
and drawbacks, not the least of which is that they require users to generate
large amounts of the compound of interest.

Now, a new, one-step imaging technique that analyzes bacterial cell shape
could enable researchers to more rapidly identify the mechanisms by which
compounds kill bacteria. The approach, described online September 17 in
Proceedings of the National Academy of Sciences, will allow industry and
academic scientists to pick out new drug candidates based on the pathways
they target or uncover new mechanisms of action to pursue in antibiotic
development as bacteria develop resistance to existing treatments [1].

Showing that a particular drug kills bacteria is not difficult, but learning
how exactly an antibiotic works is a slow and laborious process, noted
senior author Joseph Pogliano, a molecular biologist at the University of
California, San Diego.

Pogliano and his long-time collaborator Kit Pogliano (who is also his wife)
were applying antibiotics to bacteria to better understand how they grow and
divide. They wound up learning more about the antibiotics themselves and, as
a consequence, decided to refocus, applying antibiotics to E. coli
systematically and looking for differences in the cells’ appearance.

In their study, the researchers applied 41 different compounds from 26
different classes to E. coli. After staining the cultures with fluorescent
proteins and using ImageJ analysis software, they measured 14 different
parameters of morphology, including the area, perimeter, and circularity of
both the membranes and the DNA.

To their surprise, each antibiotic—even those with activity that seems
unrelated to morphology, such as inhibiting protein synthesis—affected the
cell shape in a unique way after two hours of exposure. In a double-blinded
follow-up experiment, the assay correctly assigned each of 18 antibiotics to
their targets.

The method, called “bacterial cytological profiling,” was also able to
identify the mechanism of action of spirohexenolide A, a natural product
compound that kills methicillin-resistant Staphylococcus aureus and other
species through a previously unknown mechanism. The drug’s effect on E. coli
shape was similar to that of a known drug, nisin, which kills by poking
holes in the bacterial membrane. The group confirmed the mechanism in a
follow-up study.

“Professor Pogliano’s recent paper in PNAS represents a remarkable advance in
determining the mode of action of antibacterial compounds,” noted Thomas
Keating, a principal scientist at AstraZeneca Infection Innovative
Medicines, who was not involved with the new study.

Keating, who is now collaborating with Pogliano to study the modes of action
of new compounds in E. coli and additional species, added, “His method
should prove a valuable tool in the hunt for new medicines.”

The new assay, like others already available, does not identify precise
targets—just pathways. Follow-up studies will be needed to zero in on the
molecular target, although narrowing down the list in pathways containing
only a few enzymes should be easier.

The Poglianos have founded a start-up, Linnaeus Bioscience in San Diego, to
sell bacterial cytological profiling as a service, which Pogliano says could
be used at any point in the pipeline, from a primary screen of 500,000
compounds to the lead optimization of just a handful.

“Our goal was to see that it be used as widely as possible,” Pogliano said.
“We wanted to have an avenue where it could be commercialized so that
companies could use it to find better drugs for treating
antibiotic-resistant bacteria, especially Gram negatives.”

He added that the technology will continue to improve as the group adds to its
library of cell morphology profiles. In the meantime, they are also using
the technology for their own drug discovery projects, screening natural
product libraries to find a replacement for penicillin.